During embryogenesis, the skeleton develops according to a genetic plan that controls its precise temporal and spatial formation. Heterotopic ossification results from alterations in the normal regulation of osteogenesis. Progressive osseous heteroplasia (POH) is one of the most disabling disorders of heterotopic ossification in humans. POH is a distinct autosomal dominant genetic disorder of connective tissue characterized by heterotopic ossification of skin and subcutaneous tissue during childhood with progressive and severe ossification of deep connective tissues, including tendons, ligaments and skeletal muscles, throughout life. The genetic defect and pathophysiology are unknown. Three hypotheses provide the focus for this research proposal: (1) POH is related to Albright hereditary osteodystrophy (AHO), a genetic disorder associated with minor and superficial heterotopic ossification caused by inactivating mutations in the GNAS1 gene; (2) the human GNAS1 gene is mutated in POH, resulting in reduced levels and/or activity of GNAS1 mRNA and/or protein; (3) maternal/paternal inheritance of the GNAS1 gene influences the osteogenic phenotypic of inactivating GNAS1 mutations in POH. To address these hypotheses, the applicants intend to: (1) screen genomic DNA for mutations in the GNAS1 gene in POH patients by PCR amplification and DNA sequencing (a preliminary analysis has already detected such mutations) and compare GNAS1 mutations in POH patients to those that have been found in patients with AHO; (2) examine expression of the GNAS1 gene in POH patients by quantitation of GNAS1 mRNA by RT-PCR, and Gsalpha protein by immunoblot analysis; (3) perform functional analysis of Gsalpha in POH patients by G-protein-mediated cAMP activity assays; (4) evaluate the effects of maternal/paternal inheritance of the GNAS1 gene in families with inheritance of POH; and (5) investigate Gsalpha protein expression in POH lesional tissue by immunohistochemical analysis. Analysis of the molecular genetics of POH is intended to contribute to the applicants' long-term goals of (a) understanding the molecular and cellular pathways of normal and disordered bone induction, and (b) designing rational molecular diagnostic and treatment strategies for a wide range of developmental disorders of the skeleton in humans. Accomplishment of these Specific Aims will provide the foundation for future work that will examine the pathways through which GNAS1 expression leads to regulation of normal and ectopic bone formation.